Cathepsin D 相互作用网络在鼻咽癌侵袭转移中的作用及分子机制研究

Previous work found that abnormal expression of cathepsin D was related to the occurrence and development of NPC. In this study, interacting protein network of cathepsin D was obtained by tandem affinity purification combined with mass spectrometry.Through cluster analysis, signaling pathways and protein interaction analysis, it was revealed that the two key interactome,cathepsin D/EGFR/Hsp90A of regulation molecular chaperones signaling pathway and cathepsin D / KRT6A / KRT17 of regulation cytoskeletal signaling pathways, are related to the invasion and metastasis of NPC. To investigate the correlation of the expression level among those interaction proteins and to determine the relationship among its expression level and clinic pathological features and clinical outcomes in NPC, Immunohistochemistry and Western blot analysis were performed to detect the expression of those interaction proteins. The intracellular location and combination with the situation of those interaction protein were investigated using immune fluorescent labeling and laser confocal analysis.Furthermore, the cell lines with over expression or down expression of cathepsin D were built by siRNA and gene transfection technology ,and then in vitro cell invasion assay and Western blotting were done to analysis the relationship among the expression of cathepsin D and invasion, metastasis, metastasis related protein and relevant signaling pathways in NPC. The effect and molecular mechanism of cathepsin D interaction networks will be clarified in invasion and metastasis of NPC .

在前期工作中发现组织蛋白酶D(cathepsin D)异常表达与NPC发生发展有关。本研究通过串联亲和纯化结合质谱技术获得cathepsin D相互作用蛋白网络;通过聚类分析、信号通路和蛋白相互作用分析发现两个关键相互作用组：cathepsin D/EGFR/Hsp90A调控分子伴侣信号通路和cathepsin D / KRT6A / KRT17调控细胞骨架信号通路，均与NPC侵袭转移有关；采用免疫组化和Western blot检测相互作用蛋白彼此之间表达水平的相关性及其与NPC临床病理特征的关系；免疫荧光标记和激光共聚焦观察相互作用蛋白在细胞内定位、结合情况；建立cathepsin D基因高表达或表达下调/缺失细胞系，分析cathepsin D表达水平对NPC细胞侵袭转移能力、转移相关蛋白及相关信号通路的影响，阐明cathepsin D相互作用蛋白网络在NPC侵袭转移中的作用及分子机制。

本项目在我们已取得较好的研究工作基础上，将cathepsin D打靶载体 pSUPER- si cathepsin D转染cathepsin D低表达低转移的6-10B NBC 细胞系，建立了稳定表达 myc-flag- cathepsin D的 6-10B- si cathepsin D cathepsin D细胞系, 采用TAP技术及二维液相色谱与质谱结合技术，高通量筛选与鉴定了 cathepsin D与 NPC侵袭转移相关的相互作用蛋白质；.运用生物信息学对相互作用蛋白进行了分析：包括聚类分析（Cluster）、功能注释(GO)、蛋白相互作用分析(PPI)、信号通路(KEGG Biocarta) 分析，绘制了 cathepsin D在NPC侵袭转移过程中调控的蛋白质分子网络图谱，获得了关键的蛋白相互作用组，并对其中的关键蛋白相互作用组：cathepsin D/EGFR/HSP90作了进一步的验证和功能研究；.采用免疫共沉淀和Western blot验证了cathepsin D相互作用蛋白EGFR/HSP90能在细胞内形成复合物；.施用免疫组化检测了正常鼻咽粘膜上皮、不同分化NPC和淋巴结转移NPC组织中cathepsin D/ EGFR/HSP90 NPC的表达情况，统计学分析了它们彼此之间表达水平的相关性及其与NPC临床病理特征之间的关系，初步阐明了cathepsin D相互作用蛋白在 NPC侵袭转移中的作用。